Ultrasound is a diagnostic imaging technique which provides a number of advantages over other diagnostic methodology. Unlike techniques such as nuclear medicine and x-rays, ultrasound does not expose the patient to potentially harmful exposures of ionizing electron radiation that can potentially damage biological materials, such as DNA, RNA, and proteins. In addition, ultrasound technology is a relatively inexpensive modality when compared to such techniques as computed tomography (CT) or magnetic resonance imaging.
The principle of ultrasound is based upon the fact that sound waves will be differentially reflected off of tissues depending upon the makeup and density of the tissue or vasculature being observed. Depending upon the tissue composition, ultrasound waves will either dissipate by absorption, penetrate through the tissue, or reflect back. Reflection, referred to as back scatter or reflectivity, is the basis for developing an ultrasound image. A transducer, which is typically capable of detecting sound waves in the range of 1 MHz to 10 MHz in clinical settings, is used to sensitively detect the returning sound waves. These waves are then integrated into an image that can be quantitated. The quantitated waves are then converted to an image of the tissue being observed.
Despite technical improvements to the ultrasound modality, the images obtained are still subject to further refinement, particularly in regards to imaging of the vasculature and tissues that are perfused with a vascular blood supply. Hence, there is a need for the formulation of agents that will aid in the visualization of the vasculature and vascular-related organs.
Vesicles are desirable as contrast agents for ultrasound because the reflection of sound at a liquid-gas interface, such as the surface of a vesicle, is extremely efficient.
To be effective as ultrasound contrast agents, the vesicles should be as large and elastic as possible since both these properties (bubble size and elasticity) are important in maximizing the reflectivity of sound from the vesicles. Additionally, the vesicles should be stable to pressure, i.e. retain more than 50% of the gas content after exposure to pressure. It is also highly desirable that the vesicles should re-expand after the release of pressure. Further, it is highly desirable to have a high vesicle concentration in order to maximize reflectivity and, hence, contrast. Therefore, vesicle concentration is an important factor in determining the efficacy of the vesicles. In particular, it is desirable to have more than 100.times.10.sup.6 vesicles per mL and, more preferably, more than 500.times.10.sup.6 vesicles per mL.
Size, however, remains a crucial factor in determining the suitability of vesicles for imagining. In the regime of vesicles that can pass safely through the capillary vasculature, the reflected signal (Rayleigh Scatterer) can be a function of the diameter of the vesicles raised to the sixth power so that a 4 .mu.m diameter vesicle may possess 64 times the scattering capability of a 2 .mu.m diameter vesicle.
Size is also important because vesicles larger than 10 .mu.m can be dangerous. Large vesicles have a tendency to occlude micro-vessels following intravenous or intravascular injection. Hence, it is important that the vesicles be as large as possible to efficiently reflect sound but small enough to pass through the capillaries.
In this regard, it is highly desirable that 99% of the vesicles be smaller than 10 .mu.m. Further, the mean vesicle size should be at least 0.5 .mu.m, preferably over 1 .mu.m, and more preferably close to 2 .mu.m for most effective contrast. In addition, the volume weighted mean should be on the order of 7 .mu.m.
The elasticity of the vesicles may affect their maximum permissible size since the greater the elasticity of the vesicle, the greater its ability to "squeeze" through capillaries. Unfortunately, a number of factors may prevent the formation of highly elastic vesicles, thereby further reenforcing the importance of optimizing vesicle size.
While uncoated vesicles have maximal elasticity, they are generally unstable. Consequently, efforts are often undertaken to improve the stability of the vesicles, such as by coating, that have the effect of reducing their elasticity. In addition, the use of gas or gas-precursors encapsulated in a proteinaceous shell, with the protein being cross-linked with biodegradable cross-linking agents, has been suggested, as well as the use of non-proteinaceous vesicles cross-linked covalently with biocompatible compounds. It may be assumed that such cross-linkers will add a component of rigidity to the vesicles, thus reducing their elasticity.
While it is known that liposomes can be made by shaking a solution of surfactant in a liquid medium (see, U.S. Pat. No. 4,684,479 (D'Arrigo)), a method for making vesicles having optimal size in a minimal amount of time has not heretofore been developed. Consequently, for all of the foregoing reasons, there is a need for a method and apparatus for making vesicles in which the shaking parameters are controlled so as to produce vesicles of optimum size in a minimum amount of time.